In recent years, cell surface adhesion molecules have become recognized as key mediators in numerous cellular processes including cell growth, differentiation, immune cell transmigration and response and cancer metastasis.
Four major categories of adhesion molecules have been identified: the immunoglobulin superfamily cell adhesion molecules (CAMs), cadherins, integrins and selectins.
The selectins represent a family of presently three transmembraneous, carbohydrate-binding glycoproteins: “endothelial” E-selectin, “leukocyte” L-selectin, and “platelet” P-selectin. All three selectins are divalent cation (e.g. calcium) dependent and possess an extracellular domain with a carbohydrate recognition motif, an epidermal growth factor-like motif and some smaller domains related to complement-regulatory proteins.
Human P-selectin (also referred to as GMP-140, LECAM-3, PADGEM, CD62 and CD62P) is expressed by platelets and endothelial cells. When expressed on the surfaces of these cells, its most notable effect is the slowing of leukocytes as these leave the capillaries and enter the postcapillary venules, the latter representing the major site of leukocyte-endothelium adhesion. The slowing process is observed as leukocyte rolling, signifying an initial adhesion with relatively low affinity. The firm adhesion of rolling leukocytes is primarily mediated by integrins.
In endothelial cells P-selectin is stored on Weibel-Palade bodies; in platelets it is found in the α-granules. Following activation, P-selectin is mobilized to the cell surfaces within a few minutes in response to a variety of inflammatory or thrombogenic agents. The endothelial P-selectin's primary function is to recruit leukocytes into postcapillary venules, while platelet P-selectin also results in the formation of thrombi. One of the presently known natural ligands of P-selectin is PSGL-1 (P-selectin glycoprotein ligand-1), a 120 kDa sialoprotein expressed on the surface of leukocytes where it is concentrated at the uropod. More detailed descriptions of the structure and functions of P-selectin are found in numerous publications (e.g. J. Panes, Pathophysiology 5: 271 (1999); F. Chamoun et al., Frontiers in Bioscience 5: e103 (Nov. 1, 2000) and S.-I. Hayachi, Circulation 102: 1710 (2000)).
Inflammation and inflammatory processes play a major role in the pathophysiology of numerous diseases and conditions. Conditions of the brain in which increased selectin levels were found, and which may therefore involve selectin-mediated pathophysiological events, include severe traumatic brain injury, relapsing-remitting multiple sclerosis, cerebral artery occlusion, ischemia and stroke. Conditions of the heart in which selectins are suggested to play a role include acute myocardial infarct, arterial injury, such as produced by angioplasty, and ischemia. Similarly, selectins are involved in conditions of the kidneys, such as renal injury from ischemia and reperfusion, and renal failure. Furthermore, selectins appear to play a role in organ transplant rejection, cold ischemia, hemorrhagic shock, septic shock, tumor metastasis, chronic inflammation, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, restenosis, angiogenesis, disseminated intravascular coagulation, adult respiratory stress syndrome and circulatory shock.
Thus, it would seem feasible to improve these and other conditions involving the activation of endothelial cells and leukocytes and specifically the mobilization and expression of P-selectin by specifically interrupting the P-selectin cascades. This can be done, for instance, by the administration of ligands which selectively bind to human P-selectin, but which do not possess its bioactivity. By this method mobilised P-selectin could be inactivated and leukocyte-induced tissue damage prevented. Potentially, the same effect could be achieved by gene therapy, provided the P-selectin ligand or antagonist is a peptide or modified peptide. According to this method somatic cells of a person in need of the therapy would be transfected with an expression vector carrying a DNA sequence encoding a P-selectin antagonist.
P-selectin ligands or antagonists may also be used for the prevention of diseases and conditions described above. Furthermore, such ligands may also be useful in the in vivo or in vitro diagnosis of these diseases.
Various attempts have been made in recent years to identify or create such selective ligands to P-selectin. So far, a number of substances were tested, but no clinical studies have yet provided conclusive evidence that any of these compounds produce the desired clinical effects while being tolerable in terms of side effects.
For instance, antibodies to P-selectin, that were produced and tested in animal models, were found to protect kidneys from ischemic-reperfusion injury (H. C. Rabb et al., JASN 5: 907, 1997; U.S. Pat. No. 6,033,667). In another study a recombinant soluble form of P-selectin glycoprotein ligand-1 (rPSGL-Ig) was used to inhibit thrombosis in cats (M. J. Eppihimer et al., Arteriosclerosis, Thrombosis, and Vascular Biology 20: 2483, 2000). WO-A-99/41363 discloses podocalyxin-like proteins that bind to selectins. WO-A-00/41711 describes various smaller peptides or peptide sequences that bind to members of the human selectin family; most of the sequences comprise one or more units of leucine or isoleucine.
As another approach to inhibit the P-selectin cascade various peptides derived from the lectin domain of the selectin family were found to inhibit neutrophil adhesion to P-selectin (e.g. U.S. Pat. No. 6,111,065 and U.S. Pat. No. 5,916,876); these peptides probably bind to P-selectin receptors on leukocytes.
In WO-A-94/05269 peptides are described which inhibit binding of selectins such as P-selectin, E-selectin and L-selectin. These peptides have as their core region portions of the 11-18 amino acid sequence of P-selectin, E-selectin or L-selectin. Further, WO-A-95/31210 relates to peptides and compounds that bind selectins including endothelium leukocyte adhesion molecule 1 (ELAM-1). These peptides are used for blocking adhesion of leukocytes to the selectins, i.e. especially E-selectin, but also P-selectin or L-selectin, for the purpose of inhibiting inflammation.
In Applicant's non pre-published patent application WO 03/020753 and pending international application PCT/EP03/07260 novel compounds are described with peptidic core regions and certain consensus motifs which are essential for recognising P-selectin. In contrast to most other known substances, which have an affinity to the selectins, these compounds possess a high degree of P-selectin specificity. Little is yet known about the stability of these compounds in vitro and in vivo, and—as in the case of most therapeutic or diagnostic peptides—in order to become effective it is mandatory that these compounds are injected. Most patients, however, prefer non-parenteral routes of administration, especially for chronic therapies.
Apart from the peptide approach the fact, that the selectins recognize sialyl Lewis sugar X (sLeX), has also led to a number of efforts towards the development of sugar-based ligands having selectin-binding activity. For example, U.S. Pat. No. 5,750,508 discloses synthetical ligands based on fucose and sialic acid, wherein the fucose and sialic acid moieties are separated by non-carbohydrate linkers. WO-A-96/09309 discloses oligosaccharide structures that are potential ligands to E- and P-selectin. WO-A-98/06730 describes modified oligosaccharides which are derivatives of sLeX, in which the neuraminic acid residue and the N-acetylglucosamine monomer have been replaced by various substituents. U.S. Pat. No. 5,830,871 discloses sugar-based selectin inhibitors and sLeX mimetics which can be prepared by an aldol addition reaction between a glycoside aldehyde precursor and dihydroxyacetone phosphate or a derivative thereof. However, these compounds do not have much selectivity for P-selectin.
Thus there is a need for substances with selective affinity to P-selectin, which can be used for preparing pharmaceutical compositions for the diagnosis, prevention and treatment of various diseases and conditions involving the adherence of leukocytes to vascular endothelial cells or to platelets. There is also a need for P-selectin ligands, which can be used as targeting molecules or moieties in pharmaceutical compositions for the targeting of drugs or genetic material to tissues expressing P-selectin. Furthermore, there is a need for compositions comprising said substances in a form suitable to be used in a more patient friendly way than by parenteral application.